Post-traumatic stress disorder (PTSD) is classified as a trauma- or stressor-related disorder, and evidence confirms an association between PTSD and the development of an alcohol use disorder (AUD). Despite heterogeneity in the incidence of PTSD, high comorbidity of PTSD and AUD are reported in the veteran population. Furthermore, the exacerbation of symptoms in veterans with comorbid PTSD/AUD negatively influences recovery prognosis and effective therapeutic strategies. We and others found that exposure to predator odor stress (PS), used as an animal model of PTSD, significantly increases anxiety-related behaviors and subsequent alcohol (ethanol) intake in rodents. Most importantly, we observed heterogeneity in these behavioral responses, as is observed in human PTSD/AUD patients. Additionally, prior binge drinking (BD) produced a greater enhancement in ethanol drinking following intermittent PS in stress ?sensitive? mice. Specifically, ?PS-sensitive? male and female C57BL/6J (B6) mice exhibited > 70% increase in ethanol intake (> 2.5 g/kg over baseline), while ethanol intake was unchanged in ?resilient? mice. Additional studies in B6 mice determined that BD alone produced sexually divergent changes in signaling pathways in the nucleus accumbens (NAC), while the enhanced ethanol intake following BD and PS produced sex differences in proteins related to the stress axis and neurosteroid synthesis in prefrontal cortex and hippocampus (NAC not examined in that study). In conjunction with evidence for separate behavioral and epigenetic adaptations underlying stress ?sensitivity? and ?resilience?, we hypothesize that: (1) distinct behavioral and molecular adaptations confer ?sensitivity? and ?resilience? to the enhancing effect of BD and PS on later ethanol drinking, (2) BD induces DNA methylation (DNAm) signals and associated changes in gene expression that alter adaptability to PS, and (3) sex differences exist in the underlying mechanisms in B6 mice. Aim 1 studies will determine whether ?sensitivity? vs ?resilience? to PS-enhanced drinking after prior BD is associated with altered anxiety-related behavior, heart rate (HR), and/or ethanol reward and whether sex differences exist in these relationships. Four groups will be tested: Group 1 (BD+4 weeks 23 h ethanol drinking with intermittent PS; divided into ?sensitive? and ?resilient? subgroups); Group 2 (intermittent PS only); Group 3 (nave); Group 4 (BD only). After the post-PS drinking (Group 1), final PS (Group 2), or equivalent time point (Groups 3 & 4), HR and anxiety-related behavior will be assessed in one study and conditioned place preference will be tested in a separate study. Aim 2 will identify epigenetic signals and linked gene expression profiles in the NAC that confer ?sensitivity? and ?resilience? to PS-enhanced drinking after prior BD. Groups will be as in Aim 1, but mice will be euthanized following final BD, post-PS drinking, or final PS. We predict that the genome-wide DNAm analysis will identify differentially methylated cytosines and regions mapping to novel and mostly distinct but some shared genes, regulatory regions, and networks in males and females that will be associated with ?sensitivity? and ?resilience? to PS-enhanced drinking. DNAm and gene expression association analysis will be integrated with behavioral outcomes from Aim 1 to pinpoint targets for functional analysis. Aim 3 will confirm the functional role of selected genes (Aim 2) important for ?sensitivity? and ?resilience? to PS-enhanced drinking and associated behaviors. Protein levels will be confirmed by Western blots. Pharmacological studies will manipulate candidate molecules and determine the effect on PS-enhanced drinking, BD, anxiety, and HR, beginning with 3 molecules identified during current funding. Elucidating biological and molecular mechanisms that confer ?sensitivity? and ?resilience? to escalated ethanol drinking following BD and traumatic stress can guide pharmacological treatment strategies for AUD and PTSD, which likely differ in males and females and represent important areas of focus in the effective treatment of our veterans.